Poster at the 40th annual meeting of the Biopysical Society . Abstract: Biophys. J. 70, A427 Minimal cellular phototoxicity is anticipated during two photon excitation (TPE) in fluorescence microscopy since the molecular excitation is confined to the imaged focal plane; in all other methods excitation occurs throughout the specimen. Although this advantage is being realized in practice, especially for UV absorbing fluorophores under the relatively benign conditions empirically selected for imaging, little is known about the limitations and possible damage mechanisms of this process. Parameters include excitation wavelength, excitation intensity, fluorophore selection, spatial and temporal exposures and total dose. We are measuring the inhibition of DNA synthesis by TPE in cultured HeLa cells. After exposures of controlled doses of 700 nm pulsed illumination from a mode-locked Ti:sapphire laser in an imaging raster pattern, cell proliferation is assayed by monitoring the incorporation of bromodeoxyuridine into cellular DNA via immunofluorescence labeling. Doses (W2s) are defined as the square of peak pulse power multiplied by the total illumination time in an average cell. The typical dose to form a ratio image of cellular calcium using Indo-1 fluorescence is approximately 0.05 W2s at an average power of 5 mW. The corresponding doses for the onset of DNA synthesis inhibition exceeds about1 W2s at average powers between 5 and 25 mW with 150 to 200 fs pulses. Intensity and dose dependence as well as action spectra will be presented. Supported at Developmental Resource for Biophysical Imaging and Opto-electronics by the NIH (RR04224 and RRO7719) and NSF (BIR 9419978).